CA2377458A1 - Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid - Google Patents
Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid Download PDFInfo
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Abstract
The invention relates to nucleotide sequences of four genes (folE, folP, folBand folK) from Corynebacterium glutamicumfor the biosynthesis of folic acid and their use for the microbial production of fol ic acid.
Description
GENES FROM CORYNEHACTERIUM GLUTAMICUM FOR THE BIOSYNTHESIS
OF FOLIC ACID AND THEIR USE FOR THE MICROBIAL PRODUCTION
OF FOLIC ACID
The present invention is concerned with the process for producing folic acid by fermentation using a geneti-cally manipulated organism. This invention consists of the nucleotide sequences of four genes (folE', folP, folB and folK) from Corynebacterium glutamicum for folic acid 3~iosynthesis and the use thereof for the microbial production of. folic acid. These four genes form an operon and are transcribed in the following sequence : folE, folP, folB, folK.
Folic acid is essential for animal organisms. Its derivative tetrahydrofolate is a very versatile carrier of activated one-carbon units in cells of the animal organism. Folic acid consists of three groups: a sub stituted pteridine ring, p-aminobenzoate and glutamate.
Mammals are unable to synthesize a pteridine ring. They absorb folic acid from the diet and from microorganisms in their intestinal tract. Folic acid deficiency leads mainly to lesions in the mucous membranes.
The commercial importance of folic acid is in the animal feed and human food markets. Folic acid is employed mainly as a dietary supplement.
Microorganisms can be employed for the fermentative production of folic acid. They can be optimized in their efficiency of folic acid biosynthesis by genetic manipulation of the folic acid biosynthetic pathway.
Genetic manipulation means in this connection that the number of copies and/or the rate of transcription of the genes of the folic acid biosynthetic pathway i-s la increased. As a consequence thereof, the proportion of gene product increases,, and thus the intracellular enzymic activity doss too. Increased enzymic activity leads to ~n increased rate of dietary (e. g. glucose) conversion into folic acid and thus also to an increased product concentration. For genetic manipula-tion, the nucleotide sequences of the genes of the folic acid biosynthetic pathway must be identified.
This invention is concerned with four novel gene sequences for folic acid biosynthesis from Corynebacterium glutamicum and with their use for the microbial production of folic acid.
One part of the invention comprises the fo1~ gene product. SEQ ID N0. 2 describes a polypeptide sequence.
The folE gene product encodes a polypepti.de of 202 amino acids with a molecular weight of 22 029 Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 25~ of the amino acids, most suitably up to 15~ of the amino acids, in SEQ ID NO. 2 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID N0. 2.
Another part of the invention comprises the folP gene product. SEQ ID N0. 4 describes a polypeptide sequence.
The folP gene product encodes a polypeptide of 285 amino acids with a molecular weight of 29 52~ Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 40~ of the amino acids, most suitably up to 25~ of the amino acids, in SEQ ID N0. 4 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID NO. 4.
Another part of the invention comprises the folB gene product. SEQ ID NO. 6 describes a polypeptide sequence.
The folB gene product encodes a polypeptide of 131 amino acids with a molecular weight of 14 020 Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 30~ of the amino acids, most suitably up to 20~ of the amino acids, in SEQ ID N0. 6 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID N0. 6.
Another part of the invention comprises the folK gene product. SEQ ID N0. 8 describes a polypeptide sequence.
The folFC gene product encodes a polypeptide of 160 amino acids with a molecular weight of 18 043 Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 40~ of the amino acids, most suitably up to 30~ of the amino acids, in SEQ ID N0. 8 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID N0. 8.
Another part of the invention comprises the polynucleo-tide sequences which encode the polypeptides described above. The polynucleotide sequences can be generated starting from sequences isolated from Corynebacterium glutamicum (i.e. SEQ ID NaS. 1, 3, 5 and 7) in which these sequences are modified by site-directed mutagenesis or a total chemical synthesis is carried out after back-translation of the corresponding oo9moooas - 4 -polypeptide using the genetic code.
These polynucleotide sequences can preferably be employed for the transformation of host organisms, and in this connection preferably of microorganisms, specifically in the form of gene constructs which comprise at least one copy of one of these polynucleo tides together with at least one regulatory sequence.
Regulatory sequences comprise promoters, terminators, enhancers and ribosome binding sites.
Preferred host organisms for transformation with these gene constructs are Corynebacterium and Bacillus species. It is also possible to employ any eukaryotic microorganism, preferably yeast strains of the genus Ashbya, Candida, Pichia, Saccharomyces and Hansenula.
Another part of the invention comprises the process for producing folic acid by cultivating a host organism which is transformed in the manner described above, and subsequently isolating the folic acid.
The processes and the procedures for cultivating micro organisms and for isolating folic acid from a microbial production are familiar to trained staff.
The invention is described. in more detail in the following examples, as is its use for the genetic manipulation of microorganisms, to increase the efficiency of folic acid synthesis.
Example 1 Construction of a genome library from Corynebacterium glutamicum ATCC 13032 DNA from the genome of Corynebacterium glutamicum ATCC 13D32 can be obtained by standard methods which have already been described, for example by J. Altenbuchner and J. Cullum (1984, Mol. Gen. Genet.
195: 234-13B). The genome library can be produced in accordance with standard protocols (e.g. Sambrook, J.
et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) with any cloning vector, e.g. pBluescript II KS- (Stratagene) or ZAP
Expresses (Stratagene). It is moreover possible to use any fragment size, preferably Sau3AI fragments with a length of 2-9 kb, which can be incorporated into cloning vectors with digested BamXI.
Examp 1 a 2 Analysis of the nucleic acid sequence of the genome library Individual E. coli clones can be selected from the genome library constructed in example 1. ~'. coli cells are cultivated by standard methods in suitable 'media (e.g. LB supplemented with 100 mg/1 ampicillin), and the plasmid DNA can then be isolated. Cloning of genome fragments from the DNA of Corynebacterium glutamicum into pBluescript II KS- (see example 1) allows the DNA
to be sequenced with the aid of the oligonucleotides 5'-AATTAACCCTCACTAAAGGG-3' and 5'-GTAATACGACTCACTATAGGGC-3'..
Example 3 Computer analysis of the sequences of the isolated nucleic acids The nucleotide sequences can be connected together for example With the aid of the BLASTX algorithm (Altschul et al. (1990) J. Mol. Biol. 215: 403-410). It is possible in this way to discover novel sequences and elucidate the function of these novel genes.
Example 4 Identification of an E. coli clone which comprises a nucleotide sequence of the gene for GTP cyclo hydrolase I (EC 3.5.4.16) Analysis of the E, coli clones as described in example 2, which was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID N0. 1.
On use of the BLASTX algorithm (see example 3?, this sequence.revealed similarity with GTP cyclohydrolases I
(FolE; EC 3.5.4.16) from various organisms. The greatest similarity was with the GTP cyclohydrolase I
(FolE) from Mycobacterium tuberculosis (NRDB 006273;
72~ agreement at the amino acid level).
Example 5 Identification of an E. cola clone which comprises a nucleotide sequence of the gene for dihydropteroate synthase (EC 2.5.1.15) Analysis of the E..coli clones as described in example 2, which was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID N0. 3.
On use of the BLASTX algorithm (see example 3), this sequence revealed similarity with dihydropteroate synthases (FolP; EC 2.5.1.15) from various organisms.
The greatest similarity was with the dihydropteroate synthase (FolP) from Mycobacterium tuberculosis (NRDB 006274; 53~k agreement at the amino acid level).
0091/000Q3 _ 7 _ Example 6 Identification of an E. coli clone which cor~rises a nucleotide sequence of the gene for dihydroneopt-erin aldolase (EC 4.1.2.25) Analysis of the E. coli clones as described in example 2, which was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID NO. 5.
On use of the BLASTX algorithm (see example 3), this sequence revealed similarity with dihydroneopterin aldolases (FolB; EC 4.1.2.25), from various organisms.
The greatest similarity was with the dihydroneopterin aldolase (FolB) from Mycobacterium tuberculosis (NRDB 006275; 61~ agreement at the amino acid level).
Example 7 Identification of an E. coli clone which comprises a nucleotide sequence of the gene for 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3) Analysis of the E. coli clones as described in example 2, which Was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID N0. 7.
On use of the BLASTX algorithm (see example 3), this sequence revealed similarity with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinases (FolK; EC 2.7.6.3) from various organisms. The greatest similarity was with the 2-amino-4-hydroxy-6-hydroxy-methyldihydropteridine pyrophosphokinase (FolK) from Mycobacterium leprae (ENBL AL023093; 43~ agreement at the amino acid level).
Example 8 Use of the genes for GTP cyclohydrolase I, for dihydropteroate synthase, for dihydroneopterin aldolase and for 2-amino-4-hydroxy-6-hydroxymethyldihydro-pteridine pyrophosphokinase from Corynebacterium g~utamicum for producing folic acid The genes for GTP cyclohydrolase I, for dihydropteroate synthase, for dihydroneopterin aldolase and for 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacteriurri glutamicum can be introduced with the aid of suitable cloning and expression systems into Corynebacterium glutamicum or into any other microorganism. Genetically manipulated microorganisms which differ from the wild-type organism in relation to the activity or the number of gene copies can be produced. These novel genetically manipulated strains can be employed for producing folic acid.
Sequence list (I) General information (1) Applicant:
(A) Name: BASF-LYNX Bioscience AG
(B) Street: Im Neuenheimer Feld 515 (C) City: Heidelberg (D) Country: Germany (E) Postal code: 69120 (F) Telephone: 06221/4546 (G) Fax: 06221/454770 (2) Title: Genes from Corynebacterium glutamicum for folic acid biosynthesis and their us.e for the microbial production of folic acid 0091/000Ø3 - 9 -(3)~ Number of sequences: 8 SEQ ID N0. l: DNA (folE) ATGAAGGAGACAACCGTGGATAACCACvCTGCAGTTCGCGAGTTCGATGAGGAGCGCGCAACAGC
TGCGATTCGTGAGTTGCTCATCGCTGTC~GTGAGGATCCAGATCGCGAAGGCCTGTTGGAAACCC
CAGCTCGAGTGGCTAGGGCGTACAAGGA.AACT'fTCGCGGGTCTGCATGAGGATCCCAGCACTGTG
CTGGAGAAGAGGTTCTCTGAGGGCCATGAAGAGTTGGTTCTGGTTCGTGAGATCCCGATTTACTC
CATGTGTGAGCACCACTTGGTGCCGTTCTTTGGCGTGGCGCAGATTGGTTACATTCCGGGTAAGT
CCGGCA,r~.GGTGACTGGCCTGTCCAAGCTGGCGCGTTTAGCGGATATGTTTGCTrIP.GCGACCTCRG
G'I"?'CAGGAGCGCTTGACCTCCCAAATTGCGGATGCTCTCGTCGAAAAGCTTGATGCCCAGGCCGT
GGCCGTGGTGATTGAAGCTGAGCACCi'GTGCATGGCCATGCCCGGAATCCGTAAGCCTGGTGCTG
TGACCACGACGTCTGCGGTGCGCGGCGGTTTTAAGR.ACp.ACCCTGCCTCCCGCGCTGa~GGTGTrC
TCCCTGATTCGGGGGCACTAA
SEQ ID N0. 2: amino acid (FolE) P'~CETTVDNf~AAVREFLE~RATAAIn~LL IAVt;EJ FDtt~GLL ~T
PARVARAYK~TFAGLFiEDi~'!'Til LEKTP S EGHEELVLVREI P I YSMCEHFi.L VFI'FGVAHIGYI PGKSGKVTGLSXLARLADI~'A3CRPQ
VQ~RLTSQ LADALVEKLDAQAVRWIEA~HLCMANRGIRKPGAVTTTSAVRGGFKNNAASRAEVF
SLiRGH
SEQ ID NO. 3: DNA (folP) ATGAACGTATCCTCTTTGACCATCCCGGGACGCTGTTTGGTCATGGGAAT'I'GTCAATGT~CACTGA
GGATTCCTTTTCGGACGGTGGCAAGT'ACATTGACGTTGATCAGGCGATCGCGCATGCCAAGGAAT
TGGTGGCTGCTGGCGCCGACATGRTTGATGTCGGCGGCGAGTCCACCCGGCCTGGGGCAGTGCGC
GTCGACGCCTCCGTGGAACGGGACCGGGTTG?GCCGGTCATTAAGGCGCTTCACGACGCCGGCAT
CC.aCACTTCCGTAGACACCATGCG~vGCCTCCGTGGCGCAGGCTGCCGCGGGCGCTGGCGTCTCCA
TGATCAACGACGTCTCTGGCGGTTTGGCTGATCCTGAGATGTTTTCTGTCATGflCGGAAGCGCAA
ATTCCCGTGTGTTTGATGCAC:'GGCGCACCCTCC.AATTCGGTGATGCCGCAGGTCAGGCAGA?'CA
CGGTGGAGACGTTGTAGCCGATGTGCACGCAGTGCTTGATGATCTTGTGGCCCC..CGCCACCGCTG
CTGGTGTGGCCGAAAACCAGATCGTGCT'TGATCCAGGTTTGGGTTTTGCCAAATCACGTGAAGAC
AACTGGCGTTtGCTGCAAGCACTGCCCGAGTT'TATTTCTGGACCTTTL'CCCATCCTGGTGGGAGC
ATCCCGGAAGCGAiTCCTGGCTGGCGT.'GCGCAAAGACCGTGGCCTAGATGTCACCCCCATTGATG
CCGACCCAGCAACCGCAGCGGTGACCGCAGTGTCTGCACATATGGGAGCATGGGGTwTGCGCGTG
CACGATGTCCCAGTATCAAGGGAGGCTGTTGATGTTGCCGCATTGTGGCGAAGTGGAGGAACTCA
CCATGGCTGA
0091/0000:3 - 10 -SEQ ID NO. 4: amino acid (FolP) t~IVSSLTIPGRCLVMGIVNVTEDSFSDG: tcYIDvDQA:.~AKEL'~rAAGADMIDVGGESTRPGAVR
VDASVER,DRWPVIKAJ~F3DAGIh3' SvDT?~tASVAQAAAGAGVSid'INDVSGGLADPEI~SVMAEAQ
I PVCLI~iWk T LQFGDAAGQADtiGGDWADVHAVLDDLVAP,ATAAGVA~~QIV'LDPGLGFAICSR~D
NWRLLQALpEFISGPFPILVGASRK.kFLAGVRXDRGLDVTPIDADPATAAtITAVSAf~IGAWGVRV
HI~VPVSRL~AVDVAAL4PRSGGTFyiG
SEQ ID N0. 5: DNA (FolB) ATGGCTGATCGTATTG.AACTTP.AAGGCCTTGAATGCTTCGGACACCACGGTGTGTTCGACTTTGA
AAAAGAGCAAGGCCAGCCCTTCATTGTGGATGTCACCTGCTGGATGGATTTCGATGCCGCAGGTG
CCAC-CGATGACCTTTCCCACACCG'CACATTACGGCC-CGTTGC~CATTGTTGGTTGCTGAA.ATCGTG
GAAGGCCCATCCAGGGATTTGATCGAGACGGTGGCCACGGAATCTGCGGATGCTGTGATGGCTAA
ATTTGATGCGCTTCATGCGGTGGAAGTAACCATCCATAAGC~CCF.AAGCACGGATCCCACGTACTT
TTGCTGACGTCGCGGTGGTTOCCCGACGTTCCAGGRAATCCt~.TGGC'I'~CTGGAAGGAGCAACGCC
TAA
i0 SEQ ID N0. 6: amino acid (FolB) MADkI ELICGL ~C FG~:r:GVFDFEICEQGQ c~F I VDV3'CWI~ FDAAGASDDLSDTVDYGALALLVAEIV
EGPSR.DL IETVATE SADAVMaXFDAL-t'.RV~VT I f:KPKA.P I PRTFADVAWARRSRKSi~A,AGRSNA
SEQ ID NO. 7: DNA (fold) ATGCATGCAGTTTTGTCCATCGGTTCCAACATGGATGATCGCTACGCGCTGCTCAACACAGTGAT
CGAGGAATTCAAAGATGAGATCGTGGCGCAGTCTGCGATCTe~.CTCAACCCCACCGTGGGGCATT'G
AGGATCAGGATGAATTCC'iCAACGCAGTGCTCGTTGTTGAGGTTGAAGAAACCCCCATCGAGTTG
CTGCGCCGTgGCCAAAAACTCGAAGAAGCCGCCGAGCGGGTCCGCGTCCGCAAATGGGGGCCACG
CACCCTCGATGTGGATATCGTGCAGATCATTAAAGATGGGGAAGAGATCCTTTCTGAGGATCCCG
AACTGACCTTGCCACACCCTTGGGCTTGGCAGCGTGCCTTCGTGTT'GATCCCTTC,~GTTGGAA~GCA
GAACCTGATGCCGTCCTGCACCGCACGACCATTGCAGAACATGTGGATAATCTTGATCCGACAGA
SEQ ID N0. 8: amino acid (folK) M~IAVLSTGSNt~DRYALLNTVIEEFKD~IVAQSAIYSTPPWGIEDQDEFLNAVLWEVE~TPIEL
LRRGQKLEEAAERVRVRKWGPRTLDVDIVQZIKDGLEZLSEDPELTLPFi~PWAWQRAFVLIPW'.,EA
E FDAVLHGT :'IAEHVDN'.,,DPTD i'FGVTKI
OF FOLIC ACID AND THEIR USE FOR THE MICROBIAL PRODUCTION
OF FOLIC ACID
The present invention is concerned with the process for producing folic acid by fermentation using a geneti-cally manipulated organism. This invention consists of the nucleotide sequences of four genes (folE', folP, folB and folK) from Corynebacterium glutamicum for folic acid 3~iosynthesis and the use thereof for the microbial production of. folic acid. These four genes form an operon and are transcribed in the following sequence : folE, folP, folB, folK.
Folic acid is essential for animal organisms. Its derivative tetrahydrofolate is a very versatile carrier of activated one-carbon units in cells of the animal organism. Folic acid consists of three groups: a sub stituted pteridine ring, p-aminobenzoate and glutamate.
Mammals are unable to synthesize a pteridine ring. They absorb folic acid from the diet and from microorganisms in their intestinal tract. Folic acid deficiency leads mainly to lesions in the mucous membranes.
The commercial importance of folic acid is in the animal feed and human food markets. Folic acid is employed mainly as a dietary supplement.
Microorganisms can be employed for the fermentative production of folic acid. They can be optimized in their efficiency of folic acid biosynthesis by genetic manipulation of the folic acid biosynthetic pathway.
Genetic manipulation means in this connection that the number of copies and/or the rate of transcription of the genes of the folic acid biosynthetic pathway i-s la increased. As a consequence thereof, the proportion of gene product increases,, and thus the intracellular enzymic activity doss too. Increased enzymic activity leads to ~n increased rate of dietary (e. g. glucose) conversion into folic acid and thus also to an increased product concentration. For genetic manipula-tion, the nucleotide sequences of the genes of the folic acid biosynthetic pathway must be identified.
This invention is concerned with four novel gene sequences for folic acid biosynthesis from Corynebacterium glutamicum and with their use for the microbial production of folic acid.
One part of the invention comprises the fo1~ gene product. SEQ ID N0. 2 describes a polypeptide sequence.
The folE gene product encodes a polypepti.de of 202 amino acids with a molecular weight of 22 029 Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 25~ of the amino acids, most suitably up to 15~ of the amino acids, in SEQ ID NO. 2 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID N0. 2.
Another part of the invention comprises the folP gene product. SEQ ID N0. 4 describes a polypeptide sequence.
The folP gene product encodes a polypeptide of 285 amino acids with a molecular weight of 29 52~ Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 40~ of the amino acids, most suitably up to 25~ of the amino acids, in SEQ ID N0. 4 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID NO. 4.
Another part of the invention comprises the folB gene product. SEQ ID NO. 6 describes a polypeptide sequence.
The folB gene product encodes a polypeptide of 131 amino acids with a molecular weight of 14 020 Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 30~ of the amino acids, most suitably up to 20~ of the amino acids, in SEQ ID N0. 6 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID N0. 6.
Another part of the invention comprises the folK gene product. SEQ ID N0. 8 describes a polypeptide sequence.
The folFC gene product encodes a polypeptide of 160 amino acids with a molecular weight of 18 043 Da. The present invention is also concerned with functional derivatives of this polypeptide obtainable by replacing one or more amino acids, preferably up to 40~ of the amino acids, most suitably up to 30~ of the amino acids, in SEQ ID N0. 8 by deletion, insertion or sub-stitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the poly-peptide having the sequence SEQ ID N0. 8.
Another part of the invention comprises the polynucleo-tide sequences which encode the polypeptides described above. The polynucleotide sequences can be generated starting from sequences isolated from Corynebacterium glutamicum (i.e. SEQ ID NaS. 1, 3, 5 and 7) in which these sequences are modified by site-directed mutagenesis or a total chemical synthesis is carried out after back-translation of the corresponding oo9moooas - 4 -polypeptide using the genetic code.
These polynucleotide sequences can preferably be employed for the transformation of host organisms, and in this connection preferably of microorganisms, specifically in the form of gene constructs which comprise at least one copy of one of these polynucleo tides together with at least one regulatory sequence.
Regulatory sequences comprise promoters, terminators, enhancers and ribosome binding sites.
Preferred host organisms for transformation with these gene constructs are Corynebacterium and Bacillus species. It is also possible to employ any eukaryotic microorganism, preferably yeast strains of the genus Ashbya, Candida, Pichia, Saccharomyces and Hansenula.
Another part of the invention comprises the process for producing folic acid by cultivating a host organism which is transformed in the manner described above, and subsequently isolating the folic acid.
The processes and the procedures for cultivating micro organisms and for isolating folic acid from a microbial production are familiar to trained staff.
The invention is described. in more detail in the following examples, as is its use for the genetic manipulation of microorganisms, to increase the efficiency of folic acid synthesis.
Example 1 Construction of a genome library from Corynebacterium glutamicum ATCC 13032 DNA from the genome of Corynebacterium glutamicum ATCC 13D32 can be obtained by standard methods which have already been described, for example by J. Altenbuchner and J. Cullum (1984, Mol. Gen. Genet.
195: 234-13B). The genome library can be produced in accordance with standard protocols (e.g. Sambrook, J.
et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) with any cloning vector, e.g. pBluescript II KS- (Stratagene) or ZAP
Expresses (Stratagene). It is moreover possible to use any fragment size, preferably Sau3AI fragments with a length of 2-9 kb, which can be incorporated into cloning vectors with digested BamXI.
Examp 1 a 2 Analysis of the nucleic acid sequence of the genome library Individual E. coli clones can be selected from the genome library constructed in example 1. ~'. coli cells are cultivated by standard methods in suitable 'media (e.g. LB supplemented with 100 mg/1 ampicillin), and the plasmid DNA can then be isolated. Cloning of genome fragments from the DNA of Corynebacterium glutamicum into pBluescript II KS- (see example 1) allows the DNA
to be sequenced with the aid of the oligonucleotides 5'-AATTAACCCTCACTAAAGGG-3' and 5'-GTAATACGACTCACTATAGGGC-3'..
Example 3 Computer analysis of the sequences of the isolated nucleic acids The nucleotide sequences can be connected together for example With the aid of the BLASTX algorithm (Altschul et al. (1990) J. Mol. Biol. 215: 403-410). It is possible in this way to discover novel sequences and elucidate the function of these novel genes.
Example 4 Identification of an E. coli clone which comprises a nucleotide sequence of the gene for GTP cyclo hydrolase I (EC 3.5.4.16) Analysis of the E, coli clones as described in example 2, which was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID N0. 1.
On use of the BLASTX algorithm (see example 3?, this sequence.revealed similarity with GTP cyclohydrolases I
(FolE; EC 3.5.4.16) from various organisms. The greatest similarity was with the GTP cyclohydrolase I
(FolE) from Mycobacterium tuberculosis (NRDB 006273;
72~ agreement at the amino acid level).
Example 5 Identification of an E. cola clone which comprises a nucleotide sequence of the gene for dihydropteroate synthase (EC 2.5.1.15) Analysis of the E..coli clones as described in example 2, which was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID N0. 3.
On use of the BLASTX algorithm (see example 3), this sequence revealed similarity with dihydropteroate synthases (FolP; EC 2.5.1.15) from various organisms.
The greatest similarity was with the dihydropteroate synthase (FolP) from Mycobacterium tuberculosis (NRDB 006274; 53~k agreement at the amino acid level).
0091/000Q3 _ 7 _ Example 6 Identification of an E. coli clone which cor~rises a nucleotide sequence of the gene for dihydroneopt-erin aldolase (EC 4.1.2.25) Analysis of the E. coli clones as described in example 2, which was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID NO. 5.
On use of the BLASTX algorithm (see example 3), this sequence revealed similarity with dihydroneopterin aldolases (FolB; EC 4.1.2.25), from various organisms.
The greatest similarity was with the dihydroneopterin aldolase (FolB) from Mycobacterium tuberculosis (NRDB 006275; 61~ agreement at the amino acid level).
Example 7 Identification of an E. coli clone which comprises a nucleotide sequence of the gene for 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3) Analysis of the E. coli clones as described in example 2, which Was followed by analysis, as described in example 3, of the sequences obtained thereby revealed a sequence which is described by SEQ ID N0. 7.
On use of the BLASTX algorithm (see example 3), this sequence revealed similarity with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinases (FolK; EC 2.7.6.3) from various organisms. The greatest similarity was with the 2-amino-4-hydroxy-6-hydroxy-methyldihydropteridine pyrophosphokinase (FolK) from Mycobacterium leprae (ENBL AL023093; 43~ agreement at the amino acid level).
Example 8 Use of the genes for GTP cyclohydrolase I, for dihydropteroate synthase, for dihydroneopterin aldolase and for 2-amino-4-hydroxy-6-hydroxymethyldihydro-pteridine pyrophosphokinase from Corynebacterium g~utamicum for producing folic acid The genes for GTP cyclohydrolase I, for dihydropteroate synthase, for dihydroneopterin aldolase and for 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacteriurri glutamicum can be introduced with the aid of suitable cloning and expression systems into Corynebacterium glutamicum or into any other microorganism. Genetically manipulated microorganisms which differ from the wild-type organism in relation to the activity or the number of gene copies can be produced. These novel genetically manipulated strains can be employed for producing folic acid.
Sequence list (I) General information (1) Applicant:
(A) Name: BASF-LYNX Bioscience AG
(B) Street: Im Neuenheimer Feld 515 (C) City: Heidelberg (D) Country: Germany (E) Postal code: 69120 (F) Telephone: 06221/4546 (G) Fax: 06221/454770 (2) Title: Genes from Corynebacterium glutamicum for folic acid biosynthesis and their us.e for the microbial production of folic acid 0091/000Ø3 - 9 -(3)~ Number of sequences: 8 SEQ ID N0. l: DNA (folE) ATGAAGGAGACAACCGTGGATAACCACvCTGCAGTTCGCGAGTTCGATGAGGAGCGCGCAACAGC
TGCGATTCGTGAGTTGCTCATCGCTGTC~GTGAGGATCCAGATCGCGAAGGCCTGTTGGAAACCC
CAGCTCGAGTGGCTAGGGCGTACAAGGA.AACT'fTCGCGGGTCTGCATGAGGATCCCAGCACTGTG
CTGGAGAAGAGGTTCTCTGAGGGCCATGAAGAGTTGGTTCTGGTTCGTGAGATCCCGATTTACTC
CATGTGTGAGCACCACTTGGTGCCGTTCTTTGGCGTGGCGCAGATTGGTTACATTCCGGGTAAGT
CCGGCA,r~.GGTGACTGGCCTGTCCAAGCTGGCGCGTTTAGCGGATATGTTTGCTrIP.GCGACCTCRG
G'I"?'CAGGAGCGCTTGACCTCCCAAATTGCGGATGCTCTCGTCGAAAAGCTTGATGCCCAGGCCGT
GGCCGTGGTGATTGAAGCTGAGCACCi'GTGCATGGCCATGCCCGGAATCCGTAAGCCTGGTGCTG
TGACCACGACGTCTGCGGTGCGCGGCGGTTTTAAGR.ACp.ACCCTGCCTCCCGCGCTGa~GGTGTrC
TCCCTGATTCGGGGGCACTAA
SEQ ID N0. 2: amino acid (FolE) P'~CETTVDNf~AAVREFLE~RATAAIn~LL IAVt;EJ FDtt~GLL ~T
PARVARAYK~TFAGLFiEDi~'!'Til LEKTP S EGHEELVLVREI P I YSMCEHFi.L VFI'FGVAHIGYI PGKSGKVTGLSXLARLADI~'A3CRPQ
VQ~RLTSQ LADALVEKLDAQAVRWIEA~HLCMANRGIRKPGAVTTTSAVRGGFKNNAASRAEVF
SLiRGH
SEQ ID NO. 3: DNA (folP) ATGAACGTATCCTCTTTGACCATCCCGGGACGCTGTTTGGTCATGGGAAT'I'GTCAATGT~CACTGA
GGATTCCTTTTCGGACGGTGGCAAGT'ACATTGACGTTGATCAGGCGATCGCGCATGCCAAGGAAT
TGGTGGCTGCTGGCGCCGACATGRTTGATGTCGGCGGCGAGTCCACCCGGCCTGGGGCAGTGCGC
GTCGACGCCTCCGTGGAACGGGACCGGGTTG?GCCGGTCATTAAGGCGCTTCACGACGCCGGCAT
CC.aCACTTCCGTAGACACCATGCG~vGCCTCCGTGGCGCAGGCTGCCGCGGGCGCTGGCGTCTCCA
TGATCAACGACGTCTCTGGCGGTTTGGCTGATCCTGAGATGTTTTCTGTCATGflCGGAAGCGCAA
ATTCCCGTGTGTTTGATGCAC:'GGCGCACCCTCC.AATTCGGTGATGCCGCAGGTCAGGCAGA?'CA
CGGTGGAGACGTTGTAGCCGATGTGCACGCAGTGCTTGATGATCTTGTGGCCCC..CGCCACCGCTG
CTGGTGTGGCCGAAAACCAGATCGTGCT'TGATCCAGGTTTGGGTTTTGCCAAATCACGTGAAGAC
AACTGGCGTTtGCTGCAAGCACTGCCCGAGTT'TATTTCTGGACCTTTL'CCCATCCTGGTGGGAGC
ATCCCGGAAGCGAiTCCTGGCTGGCGT.'GCGCAAAGACCGTGGCCTAGATGTCACCCCCATTGATG
CCGACCCAGCAACCGCAGCGGTGACCGCAGTGTCTGCACATATGGGAGCATGGGGTwTGCGCGTG
CACGATGTCCCAGTATCAAGGGAGGCTGTTGATGTTGCCGCATTGTGGCGAAGTGGAGGAACTCA
CCATGGCTGA
0091/0000:3 - 10 -SEQ ID NO. 4: amino acid (FolP) t~IVSSLTIPGRCLVMGIVNVTEDSFSDG: tcYIDvDQA:.~AKEL'~rAAGADMIDVGGESTRPGAVR
VDASVER,DRWPVIKAJ~F3DAGIh3' SvDT?~tASVAQAAAGAGVSid'INDVSGGLADPEI~SVMAEAQ
I PVCLI~iWk T LQFGDAAGQADtiGGDWADVHAVLDDLVAP,ATAAGVA~~QIV'LDPGLGFAICSR~D
NWRLLQALpEFISGPFPILVGASRK.kFLAGVRXDRGLDVTPIDADPATAAtITAVSAf~IGAWGVRV
HI~VPVSRL~AVDVAAL4PRSGGTFyiG
SEQ ID N0. 5: DNA (FolB) ATGGCTGATCGTATTG.AACTTP.AAGGCCTTGAATGCTTCGGACACCACGGTGTGTTCGACTTTGA
AAAAGAGCAAGGCCAGCCCTTCATTGTGGATGTCACCTGCTGGATGGATTTCGATGCCGCAGGTG
CCAC-CGATGACCTTTCCCACACCG'CACATTACGGCC-CGTTGC~CATTGTTGGTTGCTGAA.ATCGTG
GAAGGCCCATCCAGGGATTTGATCGAGACGGTGGCCACGGAATCTGCGGATGCTGTGATGGCTAA
ATTTGATGCGCTTCATGCGGTGGAAGTAACCATCCATAAGC~CCF.AAGCACGGATCCCACGTACTT
TTGCTGACGTCGCGGTGGTTOCCCGACGTTCCAGGRAATCCt~.TGGC'I'~CTGGAAGGAGCAACGCC
TAA
i0 SEQ ID N0. 6: amino acid (FolB) MADkI ELICGL ~C FG~:r:GVFDFEICEQGQ c~F I VDV3'CWI~ FDAAGASDDLSDTVDYGALALLVAEIV
EGPSR.DL IETVATE SADAVMaXFDAL-t'.RV~VT I f:KPKA.P I PRTFADVAWARRSRKSi~A,AGRSNA
SEQ ID NO. 7: DNA (fold) ATGCATGCAGTTTTGTCCATCGGTTCCAACATGGATGATCGCTACGCGCTGCTCAACACAGTGAT
CGAGGAATTCAAAGATGAGATCGTGGCGCAGTCTGCGATCTe~.CTCAACCCCACCGTGGGGCATT'G
AGGATCAGGATGAATTCC'iCAACGCAGTGCTCGTTGTTGAGGTTGAAGAAACCCCCATCGAGTTG
CTGCGCCGTgGCCAAAAACTCGAAGAAGCCGCCGAGCGGGTCCGCGTCCGCAAATGGGGGCCACG
CACCCTCGATGTGGATATCGTGCAGATCATTAAAGATGGGGAAGAGATCCTTTCTGAGGATCCCG
AACTGACCTTGCCACACCCTTGGGCTTGGCAGCGTGCCTTCGTGTT'GATCCCTTC,~GTTGGAA~GCA
GAACCTGATGCCGTCCTGCACCGCACGACCATTGCAGAACATGTGGATAATCTTGATCCGACAGA
SEQ ID N0. 8: amino acid (folK) M~IAVLSTGSNt~DRYALLNTVIEEFKD~IVAQSAIYSTPPWGIEDQDEFLNAVLWEVE~TPIEL
LRRGQKLEEAAERVRVRKWGPRTLDVDIVQZIKDGLEZLSEDPELTLPFi~PWAWQRAFVLIPW'.,EA
E FDAVLHGT :'IAEHVDN'.,,DPTD i'FGVTKI
Claims (8)
1. A polypeptide having GTP cyclohydrolase I activity and selected from the following group:
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 2 (b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 2 (b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
2. A polypeptide having dihydropteroate synthase activity and selected from the following group:
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 4;
(b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 4;
(b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
3. A polypeptide having dihydroneopterin aldolase activity and selected from the following group:
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 6 (b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 6 (b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
4. A polypeptide having 2-amino-4-hydroxy-6-hydroxy-methyldihydropteridine pyrophosphokinase activity and selected from the following group:
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 8 (b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
(a) a polypeptide having the amino acid sequence which is described in SEQ ID NO. 8 (b) a polypeptide which is modified by comparison with that in (a) by deletion, insertion or substitution of one or more amino acids.
5. A polynucleotide which encodes a polypeptide corresponding to claim 1, 2, 3 or 4.
6. A gene construct having at least one copy of a polynucleotide corresponding to claim 5, together with at least one regulatory sequence.
7. A host organism which is transformed with a gene construct corresponding to claim 6.
8. A process for producing folic acid by cultivating a host organism corresponding to claim 7 with subsequent isolation of the folic acid.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19929363A DE19929363A1 (en) | 1999-06-25 | 1999-06-25 | New Corynebacterium glutamicum GTP cyclohydrolase I, dihydropteroate synthase, dihydroneopterin aldolase and 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine pyrophosphokinase polypeptides |
| DE19929363.5 | 1999-06-25 | ||
| PCT/EP2000/005864 WO2001000845A1 (en) | 1999-06-25 | 2000-06-23 | Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2377458A1 true CA2377458A1 (en) | 2001-01-04 |
Family
ID=7912677
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002377458A Abandoned CA2377458A1 (en) | 1999-06-25 | 2000-06-23 | Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1194565A1 (en) |
| KR (1) | KR20020026469A (en) |
| CN (1) | CN1371418A (en) |
| AU (1) | AU5978200A (en) |
| CA (1) | CA2377458A1 (en) |
| DE (1) | DE19929363A1 (en) |
| WO (1) | WO2001000845A1 (en) |
| ZA (1) | ZA200200582B (en) |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298854C (en) | 1999-07-02 | 2007-02-07 | 味之素株式会社 | DNA encoding sucrose PTS enzyme II |
| US6958228B2 (en) | 2000-08-02 | 2005-10-25 | Degussa Ag | Nucleotide sequence which code for the metH gene |
| US6942996B2 (en) | 2000-08-02 | 2005-09-13 | Degussa Ag | Isolated polynucleotide from Corynebacterium encoding a homocysteine methyltransferase |
| DE10039044A1 (en) | 2000-08-10 | 2002-02-21 | Degussa | Novel polynucleotide from Coryneform bacteria coding for lysR1 gene, useful as hybridization probe for detecting DNA coding for transcription regulator lysR1 |
| DE10039049A1 (en) | 2000-08-10 | 2002-02-21 | Degussa | Novel polynucleotide from Coryneform bacteria coding for lysR3 gene, useful as a probe for detecting DNA to isolate nucleic acids coding for transcription regulator lysR3 or for producing L-amino acids, e.g., L-lysine and L-valine |
| DE10039043A1 (en) | 2000-08-10 | 2002-02-21 | Degussa | New nucleotide sequences coding for the luxR gene |
| AU2001291658A1 (en) | 2000-08-26 | 2002-03-13 | Degussa A.G. | Nucleotide sequences which code for the ccpa2 gene |
| US6815196B2 (en) | 2000-09-02 | 2004-11-09 | Degussa Ag | Nucleotide sequences encoding o-succinylhomoserine sulfhydrylase |
| US6812016B2 (en) | 2000-09-02 | 2004-11-02 | Degussa Ag | Nucleotide sequences which code for the metY gene |
| AU2001293723A1 (en) | 2000-09-09 | 2002-03-22 | Degussa A.G. | Nucleotide sequences coding for the dep33 gene |
| US6759224B2 (en) | 2000-09-09 | 2004-07-06 | Degussa Ag | Nucleotide sequences which code for the sahH gene |
| DE10045496A1 (en) | 2000-09-14 | 2002-03-28 | Degussa | New nucleotide sequences coding for the ptsi gene |
| DE10055869A1 (en) | 2000-11-10 | 2002-05-29 | Degussa | A polynucleotide encoding the nadA gene useful for the preparation of nicotinic acid or its derivatives, as probes for discovering RNA, cDNA and DNA to isolate polynucleotides or genes which code for quinolinate synthetase A |
| DE10055870A1 (en) | 2000-11-10 | 2002-05-29 | Degussa | New nucleotide sequences coding for the nadC gene |
| EP1262541A1 (en) * | 2001-05-28 | 2002-12-04 | Stichting Top-Instituut Voedselwetenschappen | Production of bioavailable folic acid |
| US7468262B2 (en) | 2003-05-16 | 2008-12-23 | Ajinomoto Co., Inc. | Polynucleotides encoding useful polypeptides in corynebacterium glutamicum ssp. lactofermentum |
| CN1952114B (en) * | 2005-10-20 | 2010-04-14 | 浙江爱迪亚营养科技开发有限公司 | Glutamic acid corynebacterium and process for preparing nicotinamide by using same |
| CN109810991B (en) * | 2019-03-02 | 2021-11-12 | 昆明理工大学 | Application of dihydropteroate synthase gene folP |
| CN111235169A (en) * | 2020-02-03 | 2020-06-05 | 昆明理工大学 | GTP cyclohydrolase I gene folE and application thereof |
| CN112852844A (en) * | 2021-03-05 | 2021-05-28 | 昆明理工大学 | Application of hydroxymethyl dihydropterin pyrophosphokinase gene folK |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5968788A (en) * | 1995-08-28 | 1999-10-19 | Toray Industries, Inc. | Method for producing folic acid |
-
1999
- 1999-06-25 DE DE19929363A patent/DE19929363A1/en not_active Withdrawn
-
2000
- 2000-06-23 EP EP00945815A patent/EP1194565A1/en not_active Withdrawn
- 2000-06-23 KR KR1020017016565A patent/KR20020026469A/en not_active Application Discontinuation
- 2000-06-23 CN CN00812014A patent/CN1371418A/en active Pending
- 2000-06-23 AU AU59782/00A patent/AU5978200A/en not_active Abandoned
- 2000-06-23 WO PCT/EP2000/005864 patent/WO2001000845A1/en active Application Filing
- 2000-06-23 CA CA002377458A patent/CA2377458A1/en not_active Abandoned
-
2002
- 2002-01-23 ZA ZA200200582A patent/ZA200200582B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN1371418A (en) | 2002-09-25 |
| KR20020026469A (en) | 2002-04-10 |
| DE19929363A1 (en) | 2000-12-28 |
| WO2001000845A1 (en) | 2001-01-04 |
| AU5978200A (en) | 2001-01-31 |
| ZA200200582B (en) | 2003-03-26 |
| EP1194565A1 (en) | 2002-04-10 |
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